The present invention relates to methods and reagents for obtaining substantially pure products by enzymatic oxidation of the hydroxyl group at specific positions on certain pyranoses and structurally related compounds. In particular, the invention concerns improved processes and reagents for making D-glucosone from D-glucose, D-xylosone from D-xylose, 5-keto-D-fructose from L-sorbose, or a mixture of 2-keto-D-gluconic acid and D-isoascorbic acid from delta-D-gluconolactone by reacting D-glucose, D-xylose, L-sorbose or delta-D-gluconolactone, respectively, with oxygen in aqueous solution at a pH between about 4 and 7 in the presence of a pyranose-2-oxidase enzyme. The invention also concerns pyranose-2-oxidase enzymes and fungi which are sources thereof. Finally, the invention pertains to pyranosone-utilizing enzymes often found in pyranose-2-oxidase enzyme preparations obtained from certain fungal sources.
As used herein:
Glucose means D-glucose.
Glucosone means D-glucosone, also known as D-arabino-2-hexosulose.
Fructose means D-fructose.
Xylose means D-xylose.
Xylosone means D-xylosone, also known as D-threo-2-pentasulose.
Xyulose means D-xyulose, also known as D-threo-2-pentulose.
Sorbose means L-sorbose.
5-ketofructose means 5-keto-D-fructose, also known as D-threo-2,5-hexodiulose.
Delta-gluconolactone means delta-D-gluconolactone, also known as D-gluconic acid delta-lactone.
2-ketogluconic acid means 2-keto-D-gluconic acid.
Isoascorbic acid means D-isoascorbic acid, also known as D-araboascorbic acid.
Pyranose means glucose, xylose, sorbose or delta-gluconolactone.
Pyranosone refers to glucosone or xylosone.
NRRL means the culture depository of the United States Department of Agriculture Northern Regional Research Laboratory in Peoria, Ill., U.S.A. All NRRL deposits referred to herein were made under the terms of the Budapest Treaty on the International recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulations promulgated thereunder.
ATCC refers to the American Type Culture Collection culture depository located in Rockville, Md., U.S.A.
CMCC refers to the private culture collection of Cetus Corporation, 1400 Fifty-Third street, Emeryville, Calif., U.S.A.
There is commercial interest in producing substantially pure pyranosones by enzymatic conversion of corresponding pyranoses. In part, this interest has grown out of recent advances in fructose production in which glucosone is made as an intermediate. U.S. Pat. No. 4,246,347, assigned to the assignee of the present application and incorporated herein by reference, discloses a fructose-production method comprising enzymatic conversion of glucose to glucosone, followed by catalytic hydrogenation of glucosone to fructose. In order to produce fructose at a purity suitable for use as a food sweetener, it is necessary that the intermediate glucosone also be substantially pure.
Glucosone also has application in the production of mannitol and sorbitol. U.S. patent application Ser. Nos. 409,990, filed Aug. 20, 1982, now abandoned and 517,996, filed Aug. 1, 1983, entitled "Process for the Production of Mannitol and Sorbitol", also assigned to the assignee of the present application and incorporated herein by reference, describe a method of producing a mixture of mannitol and sorbitol having an exceptionally high mannitol-to-sorbitol ratio. The mixture is prepared by reducing a solution of glucosone by catalytic hydrogenation, using a nickel catalyst in the presence of hydrogen. For production of a mannitol/sorbitol mixture suitable for use as a food sweetener, substantially pure glucosone must be used.
Glucosone, being a relatively reactive compound, is also expected to be of use in other synthetic processes. For example, glucosone in aqueous solution at pH between about 4.5 and about 8.5 (preferably between pH 6.0 and 6.5) can be converted, by a novel pyranosone dehydratase enzyme described herein, to the antibiotic cortalcerone (2-hydroxy-6H-3-pyrone-2-carboxaldehyde hydrate, see Baute, et al., Phytochemistry 15, 1753-1755 (1976); Baute, et al., Phytochemistry 16, 1895-1897 (1977)).
This pyranosone dehydratase enzyme first catalyses the dehydration of two of the equilibrium forms of glucosone present in aqueous solution, alpha-D-arabino-hexosulopyranose and/or beta-D-arabino-hexosulopyranose, to 2,4-dihydroxy-6-hydroxymethyl-6H-3-pyrone (and tautomers thereof). The equilibrium forms of glucosone which are substrates for the pyranosone dehydratase both have a keto group at position 2 in the pyranose ring, an R configuration at carbon 4 in the pyranose ring (the same stereochemical configuration as at carbon 4 in D-glucose), and an axial hydrogen at carbon 3 (disposed with respect to the pyranose ring in the same way as the hydrogen at carbon 3 in D-glucose). This arrangement of keto oxygen at position 2, hydroxyl at position 4, and hydrogen at position 3 of a pyranose ring apparently provides the substrate specificity for catalytic dehydration (involving removal of the hydroxyl from position 4, and hydrogen from position 3) by pyranosone dehydratase. (A hydroxyl group at carbon 1 might also be required for substrate specificity.)
For formal names of sugars and sugar derivatives used in the present specification, reference is made to R. Shallenberger, Advanced Sugar Chemistry: Principles of Sugar Stereochemistry, AVI Publishing Company, Inc., Westport, Conn., 1982, pp. 1-28. For determination of configuration at chiral centers according to the R.S. system, reference is made to J. Roberts and M. Casserio, Basic Principles of Organic Chemistry, W. A. Benjamin, Inc., New York, N.Y., 1964, pp. 592-593.
The intermediate, 2,4-dihydroxy-6-hydroxymethyl-6H-3-pyrone is named by analogy with the formal name for cortalcerone, provided in Baute (1976), supra. The oxygen in the pyrone ring is at position 1, the keto-carbon at position 3 and the double bond between positions 4 and 5.
This intermediate has a highly strained structure. Its ring opens spontaneously in aqueous solution, forming 4-deoxy-aldehydo-D-glycero-2,3-hexadiulose and tautomers thereof. The compound, and its tautomers, can recyclize between carbons 2 and 6 to form 3-deoxy-D-glycero-pentosulopyranose, 1-carboxaldehyde. This compound, which has the required steric arrangement for catalytic action by pyranosone dehydratase (an equitorial hydroxyl group on carbon 4, an axial hydrogen on carbon 3, and a keto oxygen on carbon 2) can be catalytically dehydrated by the enzyme to remove the hydroxyl from carbon 4 and one of the hydrogens from carbon 3, to produce cortalcerone, an antibiotic.
Another use of glucosone is described in U.S. Pat. No. 4,351,902, assigned to the assignee of the present application and incorporated herein by reference. The patent describes the catalytic oxidation of glucosone by glucose-1-oxidase to form 2-ketogluconic acid, which has uses in food and other industries.
The other pyranosone which can be prepared as described herein is xylosone. Xylosone can be reduced to xyulose by known methods, particularly by hydrogenation with molecular hydrogen in the presence of a heavy metal catalyst (e.g. palladium or carbon), and by other methods, as described, for the reduction of glucosone by fructose, in U.S. Pat. No. 4,246,347 and Geigert, et al., Carbohyd. Res. 113, 159-162 (1983). Xyulose can be fermented to ethanol by common yeasts (e.g. Saccharomyces cerevisiae), which are essentially incapable of fermenting xyulose, a major by-product of biomass degradation. See Wang, et al., Biochem. and Biophys. Rsch. Commun. 94, 248-253 (1980); Chiang, et al., Appl. and Environ. Microbiol. 42, 284-289 (1981). Thus, the methods and reagents of the present invention for conversion of xylose to xylosone, without subsequent significant enzymatic reaction of the xylosone, provide one step in a process for utilizing xylose from biomass to make ethanol.
The novel pyranosone dehydratase enzyme, whose catalysis of the dehydration of glucosone to cortalcerone is described above and whose properties and isolation are further described below, also catalyzes the dehydration of xylosone in aqueous solution to compounds which would be expected to have antibiotic properties similar to cortalcerone, and potentially other properties which make them useful to the fungi which produce them, starting from xylose and passing through xylosone, under conditions of stress.
Xylosone exists in aqueous solution as an equilibrium mixture of alpha,D-threo-pentosuloplyranose and beta,D-threo-pentosulopyranose. These compounds are the analogues of the alpha and beta anomers of the form of glucosone in solution on which the pyranosone dehydratase acts, as described above. The xylosones differ from the glucosones by the absence of an hydroxymethyl group at carbon 5 in the pyranose ring. The pyranosone dehydratase dehydrates one or both equilibrium forms of xylosone to 2,4-dihydroxy-6H-3-pryone (and tautomers thereof).
2,4-dihydroxy-6H-3-pyrone is, like its analog derived from glucosone, a highly strained compound. It likely ring-opens to 4-deoxy-aldehydo-2,3-pentadiulose and tautomers thereof. The compound and any of the tautomers may be hydrated at the aldehyde group at position-1.
Because 4-deoxy-aldehydo-2,3-pentadiulose lacks a sixth carbon atom, it cannot rearrange into a form with the steric requirement, described above, for the action of pyranosone dehydratase. Consequently, the enzyme dehydrates xylosone only once.
Both 2,4-dihydroxy-6H-3-pyrone, and its 6-hydroxymethyl analog have absorbance maxima in the UV at about 265 nm. 4-deoxy-aldehydo-2,3-pentadiulose (or tautomers or aldehyde-group hydrates of the compound) and 4-deoxy-aldehydo-D-glycero-2,3-hexadiulose (or tautomers or aldehyde-group hydrates of the compound) might absorb strongly near 265 nm, but do not absorb significantly at any other wavelength between about 200 nm and about 300 nm. 3-deoxy-D-glyceropentosulopyranose, 1-carboxaldehyde (in any configuration, whether hydrated or not) does not absorb in the UV between about 200 nm and about 300 nm. Cortalcerone, however, has an absorption maximum only at about 230 nm, between 200 and 300 nm. Thus, adding pyranosone dehydratase enzyme to an aqueous solution of pure xylosone results in increasing absorption at 265 nm and no absorbtion at 230 nm as the xylosone is dehydrated to the 6H-3-pyrone. However, adding pyranosone dehydratase enzyme to an aqueous solution of pure glucosone results first in increasing absorption at 265 nm, as glucosone is dehydrated to the 6H-3-pyrone, and eventually in increasing absorption at 230 nm and decreasing absorption at 265 nm as 6H-3-pyrone rearranges to non-UV-absorbing 3-deoxy-D-glycero-pentosulopyranose,1carboxaldehyde, which in turn is dehydrated by the enzyme to cortalcerone.
The 5-ketofructose obtained in substantially pure form from sorbose, with the methods and reagents provided in the present specification, can be converted to kojic acid, 3-oxykojic acid and 5-oxymaltol. See, e.g. Sato, et al., J. Agr. Biol. Chem. 31, 877-878 (1967) and 33, 1606-1611 (1969). Kojic acid itself is an antibiotic and can be converted to the flavor-enhancing additives maltol and ethyl maltol. See Merck Index, 10th Edition, p. 764 (1983).
The uses of mixtures of 2-ketogluconic and isoascorbic acids provided from delta-gluconolactone by the methods and reagents of the present specification are set forth in U.S. Pat. No. 4,351,902, supra.
The production of pyranosones, 5-ketofructose and mixtures of 2-ketogluconic and isoascorbic acids from the corresponding pyranoses using a pyranose-2-oxidase enzyme preparation from P. obtusus has been described, e.g., Janssen and Ruelius, Biochim. et Biophys. Acta 167, 501-510 (1968). Above-mentioned U.S. Pat. Nos. 4,246,347, 4,321,323, 4,321,324 and 4,423,149 describe production of glucosone from glucose using glucose-2-oxidase enzyme preparations from various Basidiomycetes.
U.S. Pat. No. 4,423,149 is assigned to the assignee of the present application and is incorporated herein by reference. Above-mentioned U.S. Pat. Nos. 4,351,902 and 4,423,149 describe production of mixtures of 2-ketogluconic and isoascorbic acids from deltagluconolactone using glucose-2-oxidase enzyme preparations from Basidiomycetes. The prior art references in this area describe reactions using partially purified pyranose-2-oxidase (P2O) derived from Polyporous obtusus, and carried out at a preferred pH of about 7.0. For a variety of reasons which will be explored below, these reaction methods inherently produce breakdown of the pyranosone products, reducing the yield and purity. The prior art methods are also characterized generally by relatively short P2O half lives, due in part to a failure to control co-produced H.sub.2 O.sub.2 levels adequately, as will be seen below.
It is therefore one general object of the present invention to provide a method and reagent by which pyranoses can be enzymatically oxidized to the corresponding pyranosones or related compounds with substantially greater yield and purity than is obtainable by prior art methods and reagents.
A particular object of the invention is to provide a method of producing substantially pure glucosone for use in making high-purity food additive sugars such as fructose, mannitol and sorbitol.
Yet another object of the invention is to provide a relatively inexpensive and easily prepared P2O reagent which has enhanced stability under the reaction conditions employed in the method of the invention.
Providing a reagent and method which are readily adaptable to commercial-scale uses is still another object of the invention.
The method of the invention includes providing a P2O enzyme or enzyme preparation which is substantially free of measurable pyranosone-utilizing enzyme activity at a selected pH between about 4.4 and 7.0. The P2O preparation is reacted with a pyranose substrate in the presence of an H.sub.2 O.sub.2 -removing catalyst, such as catalase. A preferred H.sub.2 O.sub.2 -removing catalyst includes catalase which has been purified to remove substantially all glucose-1-oxidase activity. The reaction is performed in the presence of oxygen at the selected pH between 4.4 and 6.0.
One major pyranosone-utilizing enzyme which has been identified is referred to herein as pyranosone dehydratase (PD), so called for its ability to dehydrate pyranosones such as glucosone and xylosone. This enzyme is present in partially purified P2O enzyme preparations obtained from P. obtusus. The activity of the enzyme may be substantially eliminated, according to one aspect of the invention by (a) purifying the P2O preparation to remove PD; (b) heat-treating the P2O preparation to inactivate PD preferentially; and/or (c) carrying out the reaction at about pH 4.4.
The invention also contemplates selecting, as a source of P2O, a fungal organism which is capable of converting glucose to glucosone, evidencing a P2O enzyme, but which is incapable of converting glucose or glucosone to cortalcerone, evidencing the absence of PD. Two preferred organisms include Coriolus versicolor and Lenzites betulinus.
In one embodiment of the invention, there is provided a reagent composed of a solid support and surface-attached P2O and catalase molecules, at a P2O/catalase activity ratio of between about 10.sup.-5 to 10.sup.-3. The P2O is prepared to be free of substantially all pyranosone utilizing enzyme contaminants at a selected pH between about 4.4 and 6.0.
The invention also contemplates substantially pure P2O enzymes prepared from the above-mentioned sources, as well as cultures of fungi which produce enhanced amounts of P2O enzyme.
These and other objects and features of the invention will become more fully apparent from the following description of the invention, when read in conjunction with the accompanying drawings.